The rate of osmotic hemolysis: a relationship with membrane bilayer fluidity

A first-order semilogarithmic plot of the decrease in turbidity that takes place during hemolysis is used to define an apparent rate of hemolysis. The effect on this rate of hemolysis of various membrane modifications is studied. Triton X-100, ethanol and chlorpromazine, which dissolve into the membrane, all increase the rate of hemolysis, even though the same concentration of ethanol and chlorpromazine has been shown to decrease the osmotic fragility. Glutaraldehyde, azodicarboxylic acid-bisdimethylamide (diamide) and intracellular Ca2+ are used to produce cross-links on membrane proteins. All of these reagents decrease cell deformability but have different effects on the rate of hemolysis, with Ca2+ increasing, glutaraldehyde decreasing and diamide producing almost no effect on the rate. These modifications are also found to alter the ESR spectra of the stearic acid spin-label, 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl, which probes mobility in the hydrophobic core of the lipid bilayer. A correlation between the effect of membrane modifications on bilayer fluidity and the rate of hemolysis suggests that the rate-limiting process which determines the rate of hemolysis involves rupturing of the bilayer.